The ability of the p53 tumor suppressor to induce cell cycle arrest and cell death is closely regulated under normal conditions. The transcriptional activity of p53 is negatively controlled by murine double minute (MDM2). p53 requires the coactivator CREB-binding protein (CBP), or its structural homolog, p300, to stimulate transcription of responsive genes. Here we find that the transactivation domain of p53 selectively interacts with the N- and C-terminal regions of CBP/p300. A mutant CBP lacking the N terminus failed to stimulate p53-dependent transactivation. In both p53 null Saos2 cells, and in UV-irradiated MCF7 cells, we observed that MDM2 associates with the N-terminal region of CBP/p300. Because p53 interacts with both MDM2 and CBP/p300 through its trans-activation domain, we examined the role of MDM2 in p53-coactivator interactions. MDM2 blocked CBP/p300 recruitment in vitro and inhibited the interaction of the transactivating region of p53 with both the N- or C-terminal regions of CBP/p300 in a mammalian two-hybrid assay. These observations suggest that MDM2 may be inhibiting p53 trans-activation by shielding its activation domain from the coactivators, a new mechanism for the inhibition of p53-dependent gene expression.